MI-30-B AS A HOUSEKEEPING GENE POR MIRNA QPCR QUANTIFICATION IN CHRONIC MYELOID LEUKEMIA

FREUE, JM; BIANCHINI, M; SANCHEZ, MB; RUIZ, MS

Congreso; 26th Congress of the European Hematology Association (virtual edition).; 2021

European Hematology Association

Background: Quantitative polymerase chain reaction (qPCR) isa rapid and sensitive approach to identify miRNA expression. However, because of the shorter PCR products, very few reference genes have been identified for the quantitive analysis of miRNA. Differential internal reference genes are needed to normalize the expression of miRNA and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of qPCR of miRNA. In CML there is not a standard control gene, both U6 and SNORD95 have been evaluated with discordant results. So, it is mandatory to evaluate another housekeeping gene to use in this pathology. Aim: to evaluate miR-30-b as housekeeping gene in miRNA qPCR in CML cell line and patients' samples. Methods: mirVana miRNA isolation kit (Ambion) was used for RNA extraction from peripheral blood mononuclear cells (PBMCs) from 5 patients at diagnosis and 4 healthy donors samples (DS) and K562 CML cell line previously incubated with either imatinib 1000nM and 450nM or nilotinib 120nM or 60nM according to calculated IC50 using MTT assay. For qPCR a protocol that is designed to specifically amplify and quantify miRNAs using stem-loop primers was used. SNORD95, U6 and miR-30-b were compared. Results: in human samples U6 and miR-30-b had acceptable values to be used as control genes: standard deviation (SD) was 1.38 and 1.48 ofr U6, and 1.8 and 2.3 for miR-30 in CML samples and DS respectively; while coefficient of variation (CV) was 5.8% for U6 and 6.1% for miR-30 CML samples and 7.2% and 9.9% for DS respectively. Two-way anova comparison between CML and DS was not statistically significant neither was it comparing  each RNA group individually (p>0.05). In K562 cell line SD was 1.2 for SNORD95; 1.7 for U6; and 0.2 for miR-30. CV was 52% for SNORD95, 65% for U6 and 12% for miR-30. Tukey's multiple comparisons test of each group of RNA with different treatment showed statistical significance difference (p<0.001). Summary/Conclusion: In conclusion, miR-30 is a suitable  control gene  when evaluating miRNA expression in K562 cell line and for human samples. To confirm these findings,  a bigger cohort should be evaluated. U6 was a good control gene in human samples but not in CML cell line.